ABSTRACT The process in which ubiquitin (Ub) conjugation is required for trafficking of integral membrane proteins into multivesicular bodies (MVBs) and eventual degradation in the lumen of lysosomes/vacuoles is well defined. However, Ub-independent pathways into MVBs are less understood. To better understand this process, we have further characterized the membrane protein Sna3, the prototypical Ub-independent cargo protein sorted through the MVB pathway in yeast. We show that Sna3 trafficking to the vacuole is critically dependent on Rsp5 ligase activity and ubiquitination. We find Sna3 undergoes Ub-dependent MVB sorting by either becoming ubiquitinated itself or associating with other ubiquitinated membrane protein substrates. In addition, our functional studies support a role for Sna3 as an adaptor protein that recruits Rsp5 to cargo such as the methionine transporter Mup1, resulting in efficient Mup1 delivery to the vacuole.

[Show abstract][Hide abstract]ABSTRACT:
The intracellular pathogen, Legionella pneumophila, relies on numerous secreted effector proteins to manipulate host endomembrane trafficking events during pathogenesis, thereby preventing fusion of the bacteria-laden phagosome with host endolysosomal compartments, and thus escaping degradation. Upon expression in the surrogate eukaryotic model Saccharomyces cerevisiae, we find that the L. pneumophila LegC7/YlfA effector protein disrupts the delivery of both biosynthetic and endocytic cargo to the yeast vacuole. We demonstrate that the effects of LegC7 are specific to the endosome:vacuole delivery pathways; LegC7 expression does not disrupt other known vacuole-directed pathways. Deletions of the ESCRT-0 complex member, VPS27, provide resistance to the LegC7 toxicity, providing a possible target for LegC7 function in vivo. Furthermore, a single amino acid substitution in LegC7 abrogates both its toxicity and ability to alter endosomal traffic in vivo, thereby identifying a critical functional domain. LegC7 likely inhibits endosomal trafficking during L. pneumophila pathogenesis to prevent entry of the phagosome into the endosomal maturation pathway and eventual fusion with the lysosome.

[Show abstract][Hide abstract]ABSTRACT:
When ubiquitin (Ub) is attached to membrane proteins on the plasma membrane, it directs them through a series of sorting steps that culminate in their delivery to the lumen of the lysosome where they undergo complete proteolysis. Ubiquitin is recognized by a series of complexes that operate at a number of vesicle transport steps. Ubiquitin serves as a sorting signal for internalization at the plasma membrane and is the major signal for incorporation into intraluminal vesicles of multivesicular late endosomes. The sorting machineries that catalyze these steps can bind Ub via a variety of Ub-binding domains. At the same time, many of these complexes are themselves ubiquitinated, thus providing a plethora of potential mechanisms to regulate their activity. Here we provide an overview of how membrane proteins are selected for ubiquitination and deubiquitination within the endocytic pathway and how that ubiquitin signal is interpreted by endocytic sorting machineries.

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α-synuclein (α-syn) is a small lipid binding protein implicated in several neurodegenerative diseases, including Parkinson's disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell-types from α-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase, Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and ER-to-Golgi vesicle trafficking.

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Sna3 is an Rsp5 Adaptor Protein that Relies on Ubiquitinationfor its MVB SortingChris Macdonald, Daniel K. Stringer*, and Robert C. PiperMolecular Physiology Biophysics University of Iowa, Iowa City, IA 52246AbstractThe process in which ubiquitin (Ub) conjugation is required for trafficking of integral membraneproteins into multivesicular bodies (MVBs) and eventual degradation in the lumen of lysosomes/vacuoles is well-defined. However, Ub-independent pathways into MVBs are less understood. Tobetter understand this process, we have further characterized the membrane protein Sna3, theprototypical Ub-independent cargo protein sorted through the MVB pathway in yeast. We showthat Sna3 trafficking to the vacuole is critically dependent on Rsp5 ligase activity andubiquitination. We find Sna3 undergoes Ub-dependent MVB sorting either by becomingubiquitinated itself or associating with other ubiquitinated membrane protein substrates. Inaddition, our functional studies support a role for Sna3 as an adaptor protein that recruits Rsp5 tocargo such as the methionine transporter Mup1, resulting in efficient Mup1 delivery to thevacuole.KeywordsUbiquitin; Endosome; Multivesicular body; Lysosome; VacuoleINTRODUCTIONThe levels of cell surface membrane proteins are determined in large part by their traffickingto, and degradation in, lysosomes. Integral membrane proteins destined for degradation aremodified by the attachment of ubiquitin (Ub), which serves as a signal for internalizationand subsequent entry into intralumenal vesicles (ILVs) that accumulate within lateendosomes/multivesicular bodies (MVBs). The actions of Ub ligases and deubiquitinatingenzymes, that modify proteins by adding or removing Ub respectively, dictate the fate ofproteins as they pass through the endosomal system (1, 2). Ubiquitinated cargo proteins thatmaintain a Ub signal are transported to the late endosome where the sequential action of theEndosomal Sorting Complexes Required for Transport (ESCRT) machinery incorporateubiquitinated cargo proteins into MVB ILVs (3). Many of the ESCRT subunits contain Ub-binding domains (UBDs), thus forming a system designed to process and sort ubiquitinatedcargo (4). MVBs eventually fuse with lysosomes (or in yeast, the vacuole), therebydelivering the ILV-encapsulated cargo for degradation in the lysosomal lumen.The major ligase responsible for ubiquitination of integral membrane proteins at the plasmamembrane in yeast is Rsp5 (5), which is homologous to mammalian Nedd4 (6). Thetryptophan-tryptophan (WW) domains within Rsp5 and its homologs have been shown todirectly interact with a PPXY (PY) motif (7, 8) found within some MVB cargo proteins (9,Corresponding author: robert-piper@uiowa.edu.*Present address: Laboratory of Protein Dynamics and Signaling, Center for Cancer Research, National Cancer Institute, Frederick,MD 21702NIH Public AccessAuthor ManuscriptTraffic. Author manuscript; available in PMC 2013 April 01.Published in final edited form as:Traffic. 2012 April ; 13(4): 586–598. doi:10.1111/j.1600-0854.2011.01326.x.$watermark-text$watermark-text$watermark-text

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10). However, many membrane proteins lack a PY motif and rely on interaction withadaptor proteins to recruit Rsp5 and facilitate their ubiquitination (11–14), creating aregulatory mechanism for Rsp5 specificity. Many of these Rsp5 adaptors are themselvesubiquitinated, and integral membrane Rsp5 adaptors, such as Bsd2, also become sorted intothe MVB pathway (15).One cargo protein of particular interest to trafficking studies is Sna3, a small membraneprotein that is sorted efficiently into the MVB pathway and accumulates in the vacuolelumen (16). Many studies have proposed that Sna3 does not use a Ub-dependent sortingmechanism (16–19). Studies have also revealed that Sna3 contains a PY motif that mediatesinteraction with the WW domains of Rsp5; and mutational studies disrupting this interactionhave shown it to be essential for Sna3 sorting to the vacuole (17–19). However, mutation ofthe cytosolic lysine residues within Sna3 does not perturb its vacuolar sorting, suggestingSna3 follows an Ub-independent route to the vacuole (16). In addition, cells carryingparticular mutant alleles of RSP5 that have attenuated Rsp5 Ub-ligase activity appear to sortSna3 normally through the MVB pathway to the vacuole lumen (17–20). These data supporta model whereby Sna3 accesses a Ub-independent MVB sorting route via physicalassociation with Rsp5, which in turn associates with the ESCRT apparatus (17, 18, 20, 21).This model, however, has not been fully verified since other experiments indicate that Sna3may require ubiquitination, at least to some extent, for efficient MVB sorting (18, 19).Parallels between an Ub-independent MVB sorting mechanism for Sna3 and cargoes inother systems have been drawn, since mutation of the cytoplasmic lysine residues withinseveral mammalian cargo proteins is not sufficient to fully disrupt their lysosomal entry.These include: the GPCR murine δ opioid receptor (DOR); the transferrin receptor, TfR2;and the CD4 receptor, (22–24). Where tested, each lysineless mutant cargo follows anESCRT-dependent route into MVBs (22–25). However, the exact nature of an ESCRT-dependent MVB sorting route that does not use Ub as a sorting signal has not been clarifiedyet.In this study we investigate the role ubiquitination plays in Sna3 trafficking and addressunresolved questions regarding this process. We find ubiquitination does control the sortingof Sna3 into the MVB pathway. We find that Sna3 behaves as an Rsp5 adaptor protein thatcontributes to the downregulation of the methionine transporter Mup1. In addition, ourresults indicate that sorting of Sna3 into the MVB pathway relies on ubiquitination at somelevel-whether of Sna3 itself or of substrate proteins with which Sna3 associates.RESULTSSna3 without lysine residues is sorted into the MVB pathwaySna3 containing a GFP tag readily localizes to the interior of the vacuole by following asorting route into the intralumenal vesicles of MVBs (16). Sna3 has 4 lysine residues, 2 ofwhich are predicted to be cytosolic (Figure 1A), and previous studies have shown thateliminating these lysine residues within Sna3 (in the context of a Sna3-GFP fusion) does notperturb localization to the vacuole lumen (16–18, 21). These data support the idea that Sna3need not be ubiquitinated for entry into the MVB pathway. These observations areconfirmed in Figure 1B, showing that both GFP tagged wild-type Sna3 and Sna3 lacking itscytosolic lysines (Sna3ΔK) were correctly sorted into the vacuolar lumen. In contrast, Sna3-GFP sorting into the vacuole lumen was blocked in vps4Δ cells. Vps4 is an AAA-ATPasethat is essential for MVB sorting and regulates the assembly of ESCRTs onto endosomes.Loss of Vps4 causes an accumulation of MVB cargoes on the limiting membrane ofenlarged endosomes (26). These results confirm earlier demonstrations that Sna3ΔK-GFPMacdonald et al.Page 2Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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still follows an ESCRT-dependent route into the MVB pathway and vacuolar lumen (16,20).We also used a protease-protection biochemical assay, to confirm the delivery of Sna3 intointralumenal vesicles (which are protected from proteases by the vacuolar limitingmembrane; Figure 1C). These experiments were carried out with membranes from Sna3-GFP expressing cells lacking the vacuolar hydrolase Pep4, which would otherwise degradethe intralumenal vesicles within the vacuole (27). Cell membrane fractions were incubatedwith trypsin, denatured with 5% SDS, and subjected to western blotting with anti-GFPantibodies. This revealed that ~50% of Sna3-GFP was resistant to trypsin digestion.Immunoblotting for carboxypeptidase Y (CPY), a soluble intralumenal vacuolar hydrolase,gave a similar profile, with ~50% of the protein protected from trypsin. Thus, the “latent”population of Sna3-GFP supports the microscopy result demonstrating its localization to thevacuolar lumen. The sensitivity of a portion of Sna3-GFP to trypsin digestion is likely due torupture of some of the vacuoles during the isolation of cell membranes since CPY, a well-established marker for the vacuolar lumen, had the same digestion profile. Importantly, allof the Sna3-GFP within membranes from vps4Δ mutant cells was sensitive to trypsintreatment, consistent with the accumulation of Sna3-GFP on the limiting membranes ofendosomes and vacuoles.The Sna3ΔK-GFP protein sorts normally to the vacuole lumen, however, the fused GFPretains surface-exposed lysines (15 in the GFP variant used in this study). Althougheliminating the lysines within Sna3 dramatically reduces ubiquitination of Sna3-GFP, theGFP moiety has the capacity to become ubiquitinated and it is conceivable that this lowlevel could be driving MVB sorting (19). To circumvent this problem, we fused two HAepitopes (YPYDVPDYA, which do not contain ubiquitinatable lysines) to the C-terminus ofSna3 and Sna3ΔK. Immunoblot analysis confirmed that the high molecular weight bands,corresponding to ubiquitinated forms of Sna3, were not readily formed by Sna3ΔK (Figure1D). We next assessed the distribution of Sna3-HA and Sna3ΔK-HA by trypsin protection,and found that both were protected equally well (Figure 1E), indicating that lysineubiquitination is not required for entry of Sna3 into the MVB pathway. As a further controlwe performed the protease treatment in the presence of detergent, thereby disrupting thelipid membranes and allowing the trypsin to access proteins in the vacuole. Addition ofTriton X-100 had no effect on overall Sna3 levels but led to the complete digestion of bothSna3 and CPY when trypsin is subsequently added. These findings support the model thatSna3 does not require its lysines to undergo ubiquitination for MVB sorting (17, 18, 21).However, they conflict with previous observations that used immunofluorescencetechniques to demonstrate that HA-tagged Sna3ΔK is not localized to the vacuolar lumen(19). One potential explanation for this discrepancy is that under the conditions used in theprevious studies, the intravacuolar Sna3-HA may not have been accessible for indirectimmunofluorescence detection; in yeast, this procedure typically requires harsh fixation andextraction methods (28).Sna3 sorting through the MVB pathway is dependent on both Rsp5 activity andubiquitinationWhile the above studies clearly indicate that ubiquitination of lysine residues within Sna3are not required for correct MVB sorting, there are several caveats to concluding that Sna3strictly uses a different signal than Ub for its sorting. For instance, proteins can undergoubiquitination of cysteine, serine, or threonine residues under some conditions (29). Inaddition, proteins can be ubiquitinated on their N-terminus (30) and the Sna3-associated Ub-ligase Rsp5, in particular, has been shown to be capable of ubiquitinating the N-terminus ofSna3 at least in vitro (31).Macdonald et al.Page 3Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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To test whether Sna3 sorting is completely independent of Ub, we used a recently describedtechnique whereby the catalytic domain of a deubiquitinating enzyme can be covalentlyattached to a protein of interest to make it resistant to ubiquitination (32). In addition, whenthe DUb domain is attached to the ESCRT machinery, it can block the entry into the MVBof cargo that requires cellular ubiquitination but not cargo that is fused to Ub so that the C-terminal specific DUb peptidase domain cannot remove it. Finally, when fused to the Ub-ligase Rsp5, the DUb domain dominantly inhibits Rsp5-mediated ubiquitination and cargotrafficking by acting as an antagonistic Rsp5 substrate-specific DUb. Figure 2 shows that allof these DUb fusions blocked Sna3-GFP MVB sorting, suggesting that Sna3 does rely onUb as a sorting determinant. Sna3-GFP fused to the catalytic domain of yeast Ubp7 (Sna3-GFP-Ubp7) failed to localize to the vacuole and was instead found largely at the plasmamembrane. This effect was alleviated when the catalytic cysteine residue within the Ubp7DUb domain was mutated to serine. Thus, the block imposed by the Ubp7 catalytic domainon Sna3-GFP sorting is a consequence of deubiquitination rather than of steric hindrance orthe ability of the Ubp7 domain to interact with other yeast proteins.MVB sorting of Sna3-GFP was also dramatically blocked in cells expressing Hse1-DUbindicating that Hse1-DUb is capable of removing Ub from the Sna3 cargo, effectivelyblocking its MVB sorting. The fact that Hse1 fused to catalytically inactive DUb had noeffect on Sna3 sorting confirmed that the dominant negative effect of Hse1-DUb on Sna3sorting is due to deubiquitinating activity, presumably directed towards Sna3 or a Sna3complex of proteins. Finally, expressing a dominant-negative Rsp5-DUb also disrupted thesorting of Sna3-GFP. In contrast, an Rsp5-DUb wherein the DUb domain is catalyticallyinactive did not perturb Sna3-GFP sorting into the vacuole. Previous studies have shownthat blocks in the sorting of ubiquitinated cargo imposed by DUb fusions with both ESCRTsubunits or Rsp5 can be relieved using cargo translationally fused to ubiquitin; thusdemonstrating that the MVB sorting pathway itself remains operational under theseconditions (32). If Sna3 were to use an Ub-independent sorting signal, then these proteinsshould not have blocked its transport to the MVB pathway. Taken together, our datastrongly suggest that Ub serves as a sorting signal for the entry of Sna3 into the MVBpathway and for its accumulation within the vacuole lumen.The finding that Sna3 sorting is disrupted by a fusion of Rsp5 with a DUb, supports previousstudies that demonstrate that the interaction between Rsp5 and Sna3 facilitates theubiquitination of Sna3 (17–19, 21). However, it is unclear whether the catalytic activity ofRsp5 is required or whether the association with Rsp5 is sufficient for correct Sna3 sorting(17, 18, 21). Sna3 sorting has been monitored in the context of various rsp5 mutant allelesthat have attenuated ligase activity due to point mutations in the HECT domain (Figure 3A).Most of these Rsp5 mutants had little or no effect on the MVB sorting of Sna3 (17, 18, 21).However, how well these mutations blocked Rsp5 activity is unclear, a critical problem forSna3 since it directly binds Rsp5 and qualifies as a privileged substrate that could potentiallybe ubiquitinated at a low level by even a severely compromised Rsp5. Using the recentlysolved structure of Rsp5 (33), we found these mutations map to residues buried in the Rsp5HECT domain (Fig. 3A) making it difficult to predict how they would diminish ligaseactivity, while preserving the structural integrity of Rsp5. Therefore, we used an Rsp5mutant in which the catalytic cysteine that holds the thioeseter-linked Ub prior to ubiquitinconjugation is changed to alanine. Such a conservative change of this surface exposedresidue renders HECT domains completely catalytically inactive, while still preserving theiroverall structure and in the case of Rsp5, does not alter the its level of expression (34).Figure 3B shows that the sorting of Sna3-GFP is disrupted in cells lacking Rsp5 and can berescued by reintroduction of Rsp5, a finding consistent with previous results (17). However,catalytically inactive Rsp5 with the C777A mutation was unable to facilitate Sna3 sorting toMacdonald et al.Page 4Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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the vacuole. These data suggest that the ligase function of Rsp5 is required for Sna3 to enterthe MVB pathway.Sna3 has a functional role as an Rsp5 adaptor protein for Mup1The data described above indicated that either Sna3 was ubiquitinated to produce a sortingsignal for its entry into MVBs, or a Sna3 associated protein carried the Ub signal. Althoughubiquitinated Sna3-HA was readily detected (Figure 1B), we could not detect ubiquitinatedspecies when the lysine residues were altered to arginine (Sna3ΔK-HA). Although ourexperiments cannot exclude the possibility that Sna3 is ubiquitinated on lysines at a lowlevel or from non-lysine ubiquitination, our data lend support to a model whereby anubiquitinated Sna3-associated protein works as an MVB sorting signal. Such a model wasproposed previously (35), although clear examples are lacking. This model is presented inFigure 4 and further rationalized by predicting that Sna3 may be acting as an adaptor proteinfor the Rsp5 ligase. Recently, a number of Rsp5 adaptor proteins have been identified; theseproteins contain at least one PY motif that allows them to associate with Rsp5 and otherprotein interaction domains that allow them to associate with a variety of substrate proteins.For instance, Bsd2 is an integral membrane protein with PY motifs that allows it to act as asubstrate adaptor for the metal transporter Smf1 (15). Likewise, cytosolic proteins such asBul1 and the arrestin-related trafficking adaptors (ARTs) also work as PY motif-containingadaptors for Rsp5 (11, 36–38). Sna3 contains an Rsp5-binding PY motif that is required forits MVB sorting (17–19). Thus, Sna3 may act as an Rsp5 substrate adaptor perhaps likeBsd2, which also contains two transmembrane domains. Sna3 is very abundant inintraluminal vesicles (16) and is transcriptionally activated during late log phase (39). Giventhat this is when nutrient conditions are suboptimal, it suggests Sna3 may serve particularfunctions.To test the model that Sna3 functions as an Rsp5 adaptor, we sought to find potentialmembrane proteins that could potentially be targets for this Sna3 action. A global analysis ofprotein interactions using a DHFR biomolecular complementation assay, which isparticularly suited for probing interactions between membrane proteins, demonstrated thatSna3 interacts with a variety of membrane proteins including the methionine transporter,Mup1 (40). One of the major Rsp5 adaptors for Mup1 is Art1/Ldb19, a protein that isrequired for methionine-induced ubiquitination and downregulation of Mup1 in cells grownin early/mid log phase (11). We hypothesized that Sna3 might also contribute to thedownregulation and MVB sorting of Mup1.In cells grown to log-phase in the absence of methionine where Mup1-GFP is found at theplasma membrane (Figure 5) or in mup1Δ cells (Supplementary Figure S1), Sna3-GFP islocalized to the vacuole showing that trafficking of each protein is not solely dependent onthe other. To test whether Sna3 can assist with Mup1 sorting to the vacuole, we monitoredthe internalization of Mup1-GFP induced upon addition of methionine at different timepoints in wild-type, art1Δ, and sna3Δ cells. In wild-type cells, Mup1-GFP is largely foundin the vacuole as early as 20 minutes after methionine addition. As previously demonstrated,however, in cells lacking Art1, Mup1 internalization is dramatically reduced (11), even 40minutes after methionine addition (Figure 5A). Notably, the sna3Δ mutant cells also showeda defect in Mup1 trafficking, although not as severe as that in the art1Δ cells. Despite thefact that sna3Δ cells could traffic some Mup1-GFP to the vacuole after methionine wasadded, significant amounts of Mup1-GFP remained at the cell surface 40 minutes later.When Mup1-GFP is delivered to the vacuole interior, the full-length protein is proteolyzed,generating a protease-resistant fragment of GFP. This processing of Mup1 provided anadditional way to follow the kinetics of Mup1 downregulation on sna3Δ cells (Figure 5B).Full-length Mup1-GFP contains 13 membrane-spanning domains and even optimizedMacdonald et al.Page 5Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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protocols produced aggregated Mup1 (Supplementary Figure S2), which meant full-lengthMup1-GFP was difficult to consistently analyze and quantify by SDS-PAGE. However,extraction and detection of the vacuole-processed soluble GFP (vpGFP) allowed a measureof how much Mup1-GFP was being targeted to the vacuole. In wild-type cells, methionineaddition resulted in a dramatic increase in Mup1-GFP derived vpGFP, whereas, no increasewas observed in art1Δ cells. The sna3Δ cells were also defective, producing vpGFP lessrapidly than wild-type, an effect most obvious from comparison at the 20 minute time-point.These data demonstrate that Sna3 can contribute to the downregulation and MVB sorting ofMup1.Delivery of plasma membrane proteins to the vacuole/lysosome can be induced by variousmechanisms, such as: ligand binding to pheromone receptors (41); substrate interaction withtransporters (11, 42); nutrient starvation (43) and toxic stress (44). In an effort to induceMup1 downregulation through a different mechanism, we stressed cells by growing themjust past a late log phase, at which time nutrients would be limiting. Microarray experimentshave shown that these growth conditions also induce expression of SNA3 (39). In theabsence of methionine, Mup1 was localized to the vacuole under these conditions in wild-type cells (Figure 6A). Interestingly, Mup1-GFP was also localized to the vacuole in art1Δcells, demonstrating that Art1 is not required for all modes of Mup1 downregulation. Incontrast, Mup1-GFP targeting to the vacuole was defective in cells lacking Sna3. Thesorting defect in sna3Δ cells was corrected by reintroduction of plasmid expressed Sna3-HAor Sna3ΔK-HA without lysine residues. However, sorting was not restored by Sna3ΔPY-HA,lacking the ability of binding Rsp5 (Figure 6B). These findings support the hypothesis thatSna3 not only acts as an Rsp5 adaptor protein for Mup1, but also that its role is distinct fromthat of Art1.As an additional test, we exploited our observation that a Sna3-DUb fusion protein no longerlocalizes to the vacuole (Figure 2). We reasoned that if Sna3 is a bona fide Rsp5 Ub-ligaseadaptor protein for Mup1, the expression of Sna3-DUb should disrupt the Ub-dependentsorting of target proteins such as Mup1 into the MVB pathway. We co-expressed eitherSna3-mCherry or Sna3-mCherry-Ubp7 with Mup1-GFP, and induced downregulation of cellsurface Mup1-GFP with a 40 min treatment of methionine. As expected, Sna3-mCherry andMup1-GFP were found exclusively in the vacuolar interior. However, expressing Sna3-mCherry-Ubp7 dramatically reduced the amount of Mup1-GFP delivered into the vacuole(Figure 7A). Mup1 labeling at the plasma membrane after methionine addition was morepronounced in cells expressing Sna3-GFP-Ubp7 than in sna3Δ cells (Figure 5), presumablybecause the DUb fusion dominantly acts to deubiquitinate Mup1 regardless of whether Sna3or another mechanism, such as Art1 recruitment of Rsp5, was responsible for itsubiquitination. We found that in addition to methionine inducing downregulation of Mup1,Sna3-mCherry-Ubp7 also blocked MVB sorting of Mup1-GFP in response to high celldensity (Supplementary Figure S3). Collectively, these data support a role for Sna3 as anRsp5 adaptor for Mup1.Sna3 can be sorted into the MVB pathway by its substrateAccording to our model (Figure 4), Sna3 associates with substrates such as Mup1 and usesits PY motif to bind Rsp5, which in turn ubiquitinates either Sna3 or Mup1 or both proteins.Previous studies have shown that loss of the PY motif within Sna3 (Sna3ΔPY) blocks theubiquitination and MVB sorting of Sna3 and instead causes it to localize to puncta (2–3larger puncta as well as numerous smaller puncta (17–19). We also found that Sna3ΔPY-GFP did not localize to the vacuole interior (Figure 7B) and that ubiquitination of Sna3ΔPY-HA is blocked (Figure 1D). Thus, failure of Sna3ΔPY to sort to the MVB is explained in ourmodel (Figure 4) as a consequence of loss of Ub from either Sna3 or its substrate protein.This model predicts that restoring ubiquitination of the substrate will also restore MVBMacdonald et al.Page 6Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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sorting of Sna3ΔPY. To test this, we expressed Mup1-mCherry-Ub under control of theCUP1 promoter, with the addition of copper allowing high levels of protein expression. Inaddition to the in-frame fusion of Ub, Mup1 internalization was further favored by addingmethionine to the growth media. Mup1-mCherry-Ub was exclusively found inside thevacuole. Moreover, expression of Mup1-mCherry-Ub was sufficient to also induce MVBsorting of Sna3ΔPY-GFP (Figure 7B). These data support the idea of a “piggyback”mechanism for sorting of Sna3 and its possible substrates.DISCUSSIONPrevious studies provided evidence both for and against the hypothesis that Sna3 requireslysine ubiquitination for its MVB sorting (16–19, 21). To date, this discrepancy could beexplained by constructs expressing Sna3 tagged with lysine-containing GFP that could act asacceptors for ubiquitination (17–19). By using a biochemical approach with versions ofSna3 tagged either with or without lysine residues, we have confirmed that the lysineresidues within Sna3 are not essential for its correct sorting through the MVB pathway(Figure 1).However, in contrast to previous studies that find ubiquitination superfluous for Sna3 MVBsorting, our data support the model that Ub does serve a critical role in Sna3 trafficking.This was most effectively shown by following Sna3 that was tagged with a DUb catalyticdomain that confers the ability to deubiquitinate proteins to which it is attached (32).Additionally, expressing the ESCRT-0 subunit Hse1 or the ubiquitin ligase Rsp5 as a fusionwith a DUb enzyme also disrupted Sna3 trafficking, implicating Ub as the sorting signal forESCRT mediated sorting of Sna3 into the vacuole (Figure 2). Previous data in support of aUb-independent sorting mechanism for Sna3 also included the observation that sorting wasnot perturbed in doa4Δ cells, which have reduced levels of Ub and ubiquitination (16).However, a more in-depth investigation using different strains and growing cells to differentoptical densities found that Sna3-GFP could not localize to the vacuole in some doa4Δ cells(19), supporting the view that ubiquitination was somehow important. Another indicationthat ubiquitination might play a role in this process was a kinetic delay observed in vacuolarentry of Sna3 lacking lysine residues compared to wild-type (18).A Ub-independent MVB sorting mechanism was also supported by the observation thatmutations that compromised Rsp5 activity did not block Sna3 sorting (17–20). However,Sna3 binds directly to Rsp5, likely giving Sna3 a privileged status as a substrate that mightbe effective even when Rsp5 activity is compromised. Thus, even the use of Rsp5 mutantswith residual ligase activity might be enough to support Sna3 sorting. The only previouslyused rsp5 HECT domain mutant that did not support Sna3 sorting was L733A (21), whichcould either have less ligase activity or might instead have compromised structure. We findthat direct inactivation of Rsp5 via mutation of its catalytic cysteine, while still preservingthe overall structure of Rsp5, blocks Sna3 sorting and phenocopies the rsp5Δ phenotype.This result correlates with our observation that Rsp5 fused to a DUb causes missorting ofSna3 (Figure 2). Collectively, these findings are not consistent with the view that directprotein:protein interactions via Rsp5 are what drives an apparent Ub-independent sortingroute into MVBs. In addition, these results agree with previous findings showing that properMVB sorting of Sna3ΔK-GFP in cells with reduced Rsp5 activity is severely perturbed (18).Our data (Figure 7) also agree with previous observations that mutation of the Rsp5-bindingPY motif within Sna3 ablates vacuolar localization (17–19). Yet, we were able to forceSna3ΔPY-GFP into the lumen of the vacuole by over-expressing one of its proposedsubstrate partners, Mup1, fused to Ub. This finding demonstrates that association withubiquitinated proteins is sufficient for Sna3ΔPY to be processed by the ESCRT machineryMacdonald et al.Page 7Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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and incorporated into ILVs. The idea that Sna3 can use either its own lysine ubiquitinationor piggyback with other ubiquitinated proteins for MVB sorting explains previous resultsshowing Sna3 lacking its lysine residues fails to sort correctly in mutants with reduced Rsp5activity, even though each of those mutant conditions alone does not block Sna3 sorting(18). Moreover, our data underscore the possibility that a wide variety of proteins may boosttheir MVB sorting efficiency by associating with other ubiquitinated proteins.Similar Ub-independent routes into MVBs have been proposed in mammalian cells (22, 23,35, 45, 46). Although it is important to remember that there may be other ESCRT-independent ILV pathways such as generation of exosomes, the relevant issue for ourstudies is whether sorting along an ESCRT-dependent lysosomal degradative pathway intoMVB ILVs occurs without Ub as a sorting signal. Our data suggest that the basis of usingSna3 as an example of Ub-independent MVB sorting and applying that analogy to pathwaysin higher eukaryotes may be inappropriate. Likewise, the idea that an Ub-independentmechanism for Sna3 sorting fulfills a unique requirement for formation of ILVs themselves,as proposed (47), now seems unlikely.Studies on Sna3 have thus far been focused on its ability to serve as an MVB cargo. Here wedefine a role for Sna3 as an Rsp5 adaptor for at least one substrate, the methioninetransporter Mup1. This function overlaps with other Rsp5 adaptors such as Art1, which arerequired together for optimal methionine-induced delivery of Mup1 to the vacuole. A furthertest of this adaptor model might be to analyze the ubiquitination status of Mup1 in sna3Δcells. However, in log phase cells, Sna3 only contributes to the overall efficiency of Mup1trafficking, in contrast to the necessary role Art1 plays (11). Indeed we speculate that Sna3may play its major role at later steps in the endocytic pathway after Art1 initiates Ub-dependent internalization from the plasma membrane. Regardless of exactly when Sna3 acts,it is likely to have a modest effect on the overall levels of ubiquitinated Mup1. Mup1 isknown to be ubiquitinated by other proteins such as Art1, which could operate in a variety ofcompartments other than where Sna3 may act. Experimentally, we found it impractical tomeasure such subtle differences because Mup1 readily oligomerizes once solubilized(Supplemental Figure 2). Nonetheless, the idea that Sna3 is an Rsp5 adaptor is furthersupported by the block of Mup1 trafficking to the vacuole caused by expression of Sna3-fused to a DUb as well as the ability of “pre-ubiquitinated” Mup1 (Mup1-mCherry-Ub) topull Sna3ΔPY into the MVB pathway (Figure 7).Collectively, our work indicates that Sna3 has a function as an Rsp5 adaptor protein thatfacilitates the ubiquitination of Mup1 as well as perhaps a variety of other membraneproteins. Our data demonstrate that the quondam example of Sna3 conforms to the canonicalmode whereby Ub serves as the sorting signal for ESCRT dependent MVB sorting.Moreover, it is the first to indicate that Sna3 works as an Rsp5 adaptor. Sna3 itself has beenshown to be a very abundant protein within MVB ILVs suggesting that it may facilitateubiquitination and sorting of a wide variety of proteins besides Mup1 and the few othermembrane proteins identified in interaction screens. We speculate that the abundance ofSna3, its possible wide client range, and its status as a privileged substrate for ubiquitinationby Rsp5 may help trap other ubiquitinated proteins into a membrane subdomain where theycan be largely deubiquitinated by Doa4 (thus recycling some Ub from vacuolar degradation)while collectively remaining a ubiquitinated complex of proteins sufficient for completion ofsorting into MVB ILVs.Macdonald et al.Page 8Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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Materials and MethodsReagentsGeneral chemicals and antibodies against GFP, CPY, PGK and the Hemagglutinin (HA)epitope sequence YPYDVPDYA were used as previously described (32). Yeast strains andplasmids used are listed in Tables 1 and 2 respectively.Cell CultureYeast cultures were grown in standard minimal media supplemented with appropriate aminoacids. Cells were generally grown overnight and then resuspended in fresh media and grownto mid log phase. If a specific cell density was required, optical density at 600 nm wasmonitored before cells harvesting (OD600 = 1.0, or 2.0) and further processing. Expressionof proteins under the CUP1 promoter was induced by the addition of 100 μM CuSO4 to themedia. Mup1 internalization was induced by the aseptic addition of 20 μg/ml methionine tothe media. To obtain the rsp5Δ strain used in Figure 3 we circumvented the lethalrequirement of Rsp5 by genetic modification of MGA2, which encodes one of thefunctionally redundant transcription factors required for the essential activation of the OLE1gene (48). We integrated the myc-HIS3 sequence at basepair 2196 of MGA2 to produce atruncated version of Mga2 lacking its TMD, which can function without proteasomeprocessing (49, 50). The plasmid encoding wild-type Rsp5 was then expelled from thisstrain using 5-FOA.Fluorescence microscopyCells were grown to a mid-log phase in minimal media before harvesting and resuspensionin 100 mM Tris.HCl (pH =8.0) buffer containing 0.2% (w/v) NaN3 and 0.2% (w/v) NaF.Cells expressing GFP or mCherry fluorescent fusion proteins were then spotted onto a glassplate and viewed with an epifluorescence microscope (BX60; Olympus) with a 100×objective lens with NA 1.4. Cells were viewed through either differential interferencecontrast (DIC) optics or appropriate fluorescence filters at room temperature and imageswere captured with a cooled charge-coupled device camera (Orca R2; HamamatsuPhotonics) using iVision-Mac software (Biovision Technology). Digital processing ofimages was performed in PhotoShop (version CS4; Adobe).Trypsin protection AssayOvernight cell cultures were used to inoculate 100 ml minimal media, which was grownover night to an OD600 = 1.0. Cells were then harvested and incubated in 100 mM Tris pH9.0 buffer, containing 50 mM DTT for 5 min at room temperature. Cells were harvestedagain and resuspended in 1.2 M sorbitol, 50 mM KPO4 pH 7.5, 0.05% (w/v) NaN3, 0.05%NaFl, before addition of 100 μl zymolase and incubation at 30°C for 90 min. Spheroplastswere then layered onto a 5 ml solution of 1.2 M sucrose, 50 mM KPO4 pH 7.5, 0.05% (w/v)sodium azide, 0.05% sodium fluoride in a 15 ml conical tube. Samples were centrifuged at1,000 g for 10 minutes, and supernatant discarded. Samples were then resuspended in 1 mlice cold intracellular lysis buffer (ILB; 100 mM KAc, 50 mM KCl, 200 mM sorbitol, 20mM PIPES pH 6.8) containing a protease inhibitor cocktail tablet (Roche) and stored on icefor 10 min. Sample was then centrifuged at 400 g on a tabletop centrifuge at 4°C for 10 min.The supernatant was then transferred to a fresh Beckman ultrafuge tube and subjected tohigh speed centrifugation at 40,000 rpm for 30 minutes. The pellet was resuspended in ILBlacking protease inhibitors and the relative protein concentration was determined using aBCA assay. Samples were normalized to equal protein concentrations and then separatedinto separated tubes and treated as indicated with ILB alone, 25 μg/ml trypsin, and TritonX-100 at a final concentration of 0.2% (v/v). Samples were incubated at room temperatureMacdonald et al.Page 9Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

Figure 1. Sna3 lacking cytosolic lysine residues can traffic to the vacuoleA) Schematic representation of Sna3 depicting its two predicted transmembrane domainsand the relative location of its PY motif and lysine residues.B) Localization of Sna3-GFP or Sna3ΔK-GFP in pep4Δ and pep4Δ vps4Δ cells Bar = 5μm.C) Post-nuclear supernatants were prepared from cells in A and treated in the absence ofpresence of 25 μg /ml trypsin for 15 min at 22°C before addition of protease inhibitors.Samples were then subjected to immunoblot analysis using antibodies against GFP andCPY.D) Lysates from wild-type cells expressing versions of Sna3 (wild-type, ΔK and ΔPY)tagged with a C terminal 2 x HA epitope were prepared for iummunoblot analysis usinganti-HA antibodies. High (50 μg) and low (20 μg) levels of lysates are depicted (upperpanel). * indicates ubiquitinated species. An over-exposure better illustrating ubiquitinatedbands is shown in lower panel.E) Trypsin protection experiments with post-nuclear supernatants from pep4Δ cellsexpressing HA-tagged Sna3 and Sna3ΔK. Samples were treated with or without trypsin andin the presence or absence of Triton X-100.Macdonald et al.Page 13Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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Figure 2. The ubiquitination of Sna3 is required for sorting to the vacuoleA) Localization of Sna3-GFP wild-type cells in log phase.B) Localization in wild-type cells of Sna3-GFP fused to catalytic domain of Ubp7 (left) orthe same domain rendered catalytically inactive (right).C) Localization of Sna3-GFP in wild-type cells co-expressing Hse1 fused to the catalyticdomain of the UL36 DUb (left) or the same domain rendered catalytically inactive (right).D) Localization of Sna3-GFP in wild-type cells co-expressing Rsp5 fused to active (left) orinactive (right) catalytic domain of Ubp7. Expression of Rsp5-DUb and Hse1-DUb proteinswas under the copper-inducible control for the CUP1 promoter. Cells were grown in 100μM CuSo4 for 6 hrs prior to imaging. Schematic representations of how each of thedeubiquitinating enzyme fusions are utilized in each experiment are depicted on the right.Macdonald et al.Page 14Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text

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Figure 3. Ligase activity of Rsp5 is essential for MVB sorting of Sna3A) Point mutations of Rsp5 previously used in studies on Sna3 trafficking were mapped onto the Rsp5 HECT domain crystal structure (PDB: 3OLM). The N-lobe is colored cyan andthe C-lobe is magenta. Mutant residues that did not affect Sna3 sorting are labeled in blue(G555D; (17, 20, 21), G707D, G747E, G731I and P784T (18)) and L733, that blocksvacuolar entry of Sna3-GFP when mutated to serine (21), is labeled in green. The C777Amutant that renders Rsp5 inactive for ligase activity is labeled in red. Two orientations of themolecular surface are depicted on the right with the same labeling of residues.B) Sna3-GFP localization in rsp5Δ cells and rsp5Δ cells expressing wild-type orcatalytically inactive (C777A) Rsp5.Macdonald et al.Page 15Traffic. Author manuscript; available in PMC 2013 April 01.$watermark-text$watermark-text$watermark-text